The research proposed is directed toward ultrastructural analysis of chromatin as a way to study chromosome function, with emphasis on the development of a novel approach to unresolved problems of the regulation of eukaryotic gene expression. Nondestructive electron microscopic preparative procedures will be coupled with the identification of specific chromatin-associated proteins by polymethylmethacrylate sphere-tagged antibodies in order to analyze the interaction of histone HI with interphase and mitotic chromatin. The same approach will be used to identify and analyze the distribution of eukaryotic RNA polymerases on transcribed and untranscribed chromatin. In attempts to identify specific active structural genes, the development of in situ hybridization of DNA complimentary to a specific RNA to nascent RNA chains will be pursued. These studies should provide an independent, but related, approach to the problem of the binding of proteins to chromatin and resultant specific transcription. The composition, synthesis and functional properties of kinetochores will be investigated in order to understand the role of this organelle in specific microtubule polymerization and chromosome segregation. Analysis of the interactions between kinetochore and centromeric heterochromatin components should provide insight into recognition mechanism involved in the placement of the kinetochore at the proper site on chromatin.